灰飞虱脂肪酶LsLPS的原核表达及Ni-NTA纯化  

Prokaryotic expression and Ni-NTA purification of lipase LsLPS from Lao-delphax striatellus

作　　者：王利华[1,2] 张玉[1,2] 郑州廷 曹越 纪锐 方继朝 WANG Li-hua;ZHANG Yu;ZHENG Zhou-ting;CAO Yue;JI Rui;FANG Ji-chao(College of Plant Protection,Nanjing Agricultural University,Nanjing 210095,China;Institute of Plant Protection,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)

机构地区：[1]南京农业大学植物保护学院,江苏南京210095 [2]江苏省农业科学院植物保护研究所,江苏南京210014

出　　处：《江苏农业学报》2022年第3期611-616,共6页Jiangsu Journal of Agricultural Sciences

基　　金：江苏省农业科技自主创新基金项目[CX(20)1004];国家自然科学基金项目(31572004);江苏省自然科学基金项目(BK20170072)。

摘　　要：灰飞虱是危害中国水稻的3种主要稻飞虱之一。脂肪酶是脂肪代谢的关键酶,在昆虫发育、繁殖等生理活动中具有重要作用。为了深入研究脂肪酶(LsLPS)在灰飞虱生长发育中的功能,本研究克隆了LsLPS基因ORF序列,并进行了原核表达和His标签融合蛋白的纯化。结果显示,灰飞虱LsLPS开放阅读框长1293 bp,可翻译成430个氨基酸,含有信号肽序列和PF00151结构域。LsLPS连接到原核表达载体pET28a(+)-SUMO、pCold I后在表达菌株BL21(DE3)中主要以包涵体形式表达,连接到pET43.1a(+)后可在表达菌株BL21(DE3)中可溶性表达。采用pH 8.0、含20 mmol/L咪唑的磷酸缓冲液作为平衡液,Ni-NTA纯化LsLPS-pET43.1a(+)融合蛋白的纯度和产量相对较高。Small brown planthopper(SBPH),Laodelphax striatellus,is one of the three main rice planthoppers damaging rice in China.Lipase is the key enzyme in fat metabolism and plays an important role in insect development,reproduction and other physiological activities.In order to investigate the function of lipase in the growth and development of SBPH,the ORF sequence of LsLPS gene was cloned,and the fusion protein with a His-tag was purified.The results showed that the open reading frame of LsLPS was 1293 bp in length and could be translated into 430 amino acids,containing signal peptide sequence and PF00151 domain.LsLPS fusion proteins expressed using prokaryotic expression vectors pET28a(+)-SUMO and pCold I were mainly in the form of inclusion bodies in the expression strain BL21(DE3).After connecting LsLPS with pET43.1a(+)vector,the obtained proteins were expressed in soluble forms in BL21(DE3).The purity and yield of the fusion protein purified by Ni-NTA were relatively higher using phosphate buffer(pH 8.0)containing 20 mmol/L imidazole as equilibrium solution.

关 键 词：灰飞虱 脂肪酶 LsLPS基因 原核表达 Ni-NTA纯化 

分 类 号：S435.112.3[农业科学—农业昆虫与害虫防治]